Apparatus and method for measuring concentration of an analyte in bio-samples

ABSTRACT

An apparatus for measuring a concentration of an analyte in a bio-sample using an electrochemical bio-sensor, includes a connector with a sample cell in which an oxidation/reduction enzyme and an electron transfer mediator are fixed and a working electrode and an counter electrode are provided; a digital-to-analog converter circuit configured to apply a constant DC voltage to start the oxidation/reduction reaction of the analyte, proceed with an electron transfer reaction, and apply a Λ-step ladder-type perturbation potential for fluctuating a potential of the sample cell after applying the constant DC voltage; and a microcontroller configured to control the digital-to-analog converter circuit and directly obtain a concentration value of the analyte from a calibration equation using the Λ-step ladder-type perturbation potential. The apparatus can improve measurement accuracy by effectively minimizing a matrix interference effect of a background material in a bio-sample, particularly an inaccuracy caused by a change in hematocrit.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 14/564,002,filed on Dec. 8, 2014, which claims priority to and the benefit ofKorean Patent Application No. 10-2014-0123816 filed on Sep. 17, 2014,the entirety of each of which is incorporated herein by reference forall purposes.

BACKGROUND OF THE INVENTION (a) Field of the Invention

The present invention relates to an apparatus for measuring aconcentration of an analyte in a bio-sample, and more particularly, toan apparatus for measuring a concentration of an analyte in a bio-sampleby which concentrations of blood samples can be measured with minimumdeviation caused by a hindering material by additionally applying a stepladder-type perturbation potential for a short time and optimizing afunction formed of features obtained from induced currents in regions,to which a constant voltage and the perturbation potential are applied,as a calibration equation for multivariable regression analysis withrespect to the blood samples under various conditions if there is widedeviation in measurement results due to various hindering materials ofblood, particularly, hematocrit when concentrations of the blood samplesare measured by chronoamperometry.

(b) Description of the Related Art

It is important to measure a concentration of a clinically importantmaterial for the sake of diagnosis and health care. Particularly,measurement of a concentration of a metabolite (analyte) such asglucose, ketone, creatine, lactate, neutral fat, pyruvate, alcohol,bilirubin, NAD(P)H, and uric acid from fluid in vivo such as bloodbecomes the key to diagnosis of a disease and care of symptoms of adisease.

As a method for accurately, rapidly, and economically measuring aconcentration of a clinically significant material from fluid in vivo, amethod using an electrochemical bio-sensor has been widely used.

In such an electrochemical bio-sensor (sometimes referred to as a“strip”), a pair of electrodes (a working electrode and an counterelectrode) in which a sample cell having a capillary tube structure iscoated with a reagent including an enzyme, an electron transfermediator, and various stabilizers and dispersants are disposed.

If the sample cell of the electrochemical bio-sensor is filled withblood of a user and then installed in a portable measuring apparatus, aconstant voltage is applied to the working electrode and a currentobtained from the working electrode is measured. A concentration of ananalyte calculated according to a programmed algorithm is displayed on ascreen of the portable measuring apparatus within several seconds toseveral minutes.

Measurement and monitoring of a metabolite, i.e. an analyte, using suchan electrochemical bio-sensor is fast, convenient, and inexpensive, andthus has been widely used all over the world.

However, users and health management organizations in many countrieshave demanded an electrochemical bio-sensor with accuracy as well asconvenience, and such a demand has been specified as internationalstandards such as DeletedTextsISO 15197:2013.

Hematocrit is one important factor among factors hindering accuracy ofan electrochemical bio-sensor that measures a concentration in blood.This is because movement and diffusion speed of an oxidation/reductionreaction material depends on hematocrit of a whole blood sample andgreatly affects a measured current signal.

For example, even if blood samples have the same blood glucoseconcentration, in a blood sample having a higher hematocrit, there isresistance to movement of an oxidation/reduction reaction material.Thus, a measured current signal is decreased. On the contrary, in ablood sample having a lower hematocrit, a measured current signal isincreased.

Such an increase or decrease in a current signal causes a measured bloodglucose concentration to be lower or higher than it actually is and thusmakes the measurement inaccurate. In order to correct such inaccuracy,technologies for improving accuracy by adjusting an electrochemicalreaction time to be longer or introducing an additional device into abio-sensor even if accepting an increase in cost of measurement havebeen suggested.

A method of removing red blood cells in advance with a filter and thenmeasuring an analyte in an effort to minimize a deviation caused byhematocrit has been suggested (U.S. Pat. Nos. 5,708,247 and 5,951,836).Such a method may be effective, but needs a sensor to be manufactured byadding a filter to a strip, and thus a manufacturing process may becomplicated and costs of products may be increased.

Red blood cells hinder diffusion and movement of a material in a bloodsample and thus changes resistance of blood. Thus, a method of reducinga deviation caused by hematocrit using a net structure has beensuggested (U.S. Pat. No. 5,628,890).

Further, a method in which red blood cells are hemolyzed with a reagentand hemoglobin flowing out to blood plasma subsidiarily controls anincrease/decrease in a current signal caused by a change in hematocrithas been suggested (U.S. Pat. No. 7,641,785). However, theabove-described methods are limited in effect in a wide range ofhematocrit.

Recently, a method of correcting a deviation caused by hematocrit byelectrochemically obtaining an additional signal has been suggested. Forexample, there is a method in which an AC voltage is applied andimpedance of a blood sample is measured, and after a hematocrit value ismeasured, a measurement value of an analyte is corrected using themeasured hematocrit value (U.S. Pat. No. 7,390,667 and U.S. PatentLaid-open Publication Nos. 2004-0079652, 2005-0164328, 2011-0139634, and2012-0111739).

However, such methods require application of a simple DC voltage to ameasuring apparatus to measure impedance and also require an additionalcircuit for measuring AC and impedance in addition to a currentmeasuring circuit, and a bio-sensor is provided with an additionalelectrode for measuring conductivity or impedance. Therefore, suchmethods may increase complexity and cost of the overall measuring system(U.S. Pat. No. 7,597,793 and U.S. Patent Laid-open Publication No.2011-0139634).

Further, many Patent Documents have suggested methods of obtaining aplurality of induced current values while mixing and applying aplurality of square wave voltages different from each other in level atvarious time intervals without using an AC voltage, and compensatinghematocrit based on the obtained induced current values (U.S. Pat. Nos.6,475,372 and 8,460,537, U.S. Patent Laid-open Publication No.2009-0026094, European Patent Laid-open Publication No. 2,746,759, andWO2013/164632).

These methods have a merit in that they can be applied even withoutreplacing a conventional bio-sensor or measuring apparatus. However, inthese methods, not only may a current caused by an inducedelectrochemical reaction among a material to be measured, an enzyme, andan electron transfer mediator be generated, but also a current(background current) caused by an uncontrollable electrochemicalreaction between oxidation/reduction reaction materials remaining at anelectrical double layer on an electrode surface when an applied voltageis sharply changed may be generated.

Therefore, as for bio-sensors produced under mass production, a surfacestatus of an electrode or solubility of a reagent and homogeneity of areaction in each strip sensor cannot be exactly the same, and thus it isdifficult to regulate precision of a background current generated whenan applied voltage is sharply changed within a statistical error range.Further, it is impossible to precisely regulate a charging currentgenerated when an applied voltage is sharply changed to be equivalent ineach bio-sensor electrode. Therefore, precision in correction may bedecreased.

The present inventors found that cyclic voltammetry with periodicity canbe effective in reducing a deviation with respect to hematocrit, andapplied the cyclic voltammetry together with chronoamperometry (KoreanPatent Laid-open Publication No. 2013-0131117).

As compared with other methods using various mixed square wave voltagesto correct hematocrit, this method can reduce an effect of an unstablecharging current caused by a sharp change in voltage, and while thevoltages are scanned, concentrations of oxidation/reduction reactionmaterials present within an electrical double layer on an electrodesurface are changed with an appropriate gradient of voltage comparedwith a change in voltage. Therefore, a background current to begenerated is regulated within a specific range, and thus it is possibleto improve an overall correction effect.

However, in this method, hematocrit is separately estimated usingcurrents obtained by the cyclic voltammetry, and then the estimatedhematocrit is applied to an equation for a concentration so as tocorrect an effect of hematocrit. Thus, the overall correction effectlargely depends on accuracy of the estimated hematocrit.

Further, this method may need a complicated measuring circuit ascompared with a case where only chronoamperometry with a constant squarewave voltage is used in order to stably implement the cyclic voltammetryand measure induced currents corresponding thereto.

A method of measuring a concentration of blood in which hematocrit iscorrected by acyclically applying voltages on forward and reverse scanusing acyclic voltammetry in an electrochemical bio-sensor has beensuggested (U.S. Pat. No. 8,287,717).

Likewise, in this method, hematocrit needs to be obtained byappropriately mixing induced currents formed of voltage functions whichcan be obtained by applying the acyclic voltammetry, the hematocritobtained by an additional equation needs to be applied to the equationfor a concentration of blood to remove a matrix effect, and anadditional circuit capable of responding to a rapid scan in a widevoltage range is needed.

In addition to the above-described methods, many efforts to minimize orremove an effect of hematocrit can be found. However, most of thesemethods need a new strip structure or need to use a measuring apparatusincluding an additional circuit structure, or cannot use conventionalstrips and measuring apparatuses.

The above information disclosed in this Background section is only forenhancement of understanding of the background of the invention andtherefore it may include information that does not form the prior artthat is already known in this country to a person of ordinary skill inthe art.

SUMMARY OF THE INVENTION

The present invention has been made in an effort to provide an apparatusfor measuring a concentration of an analyte in a bio-sample using anelectrochemical bio-sensor, the apparatus including:

a connector to which a sample cell in which an oxidation/reductionenzyme capable of catalyzing an oxidation/reduction reaction of theanalyte and an electron transfer mediator are fixed and a workingelectrode and an counter electrode are provided is inserted;

a digital-to-analog converter circuit configured to apply a constant DCvoltage to start the oxidation/reduction reaction of the analyte,proceed with an electron transfer reaction, and apply a Λ-stepladder-type perturbation potential for fluctuating a potential of thesample cell after applying the constant DC voltage; and

a microcontroller configured to control the digital-to-analog convertercircuit and directly obtain a concentration value of the analyte from acalibration equation using the Λ-step ladder-type perturbationpotential.

In the method and the apparatus for measuring a concentration of ananalyte in a bio-sample according to an exemplary embodiment of thepresent invention, the first or second induced current different fromeach other in characteristics with respect to a constant voltage and anapplied voltage formed of a Λ (lambda)-step ladder-type perturbationpulse (or perturbation potential) are modified to a predeterminedfeature, and a calibration equation is obtained by an appropriatestatistical mathematical method. Therefore, it is possible to measure aconcentration of the analyte material by removing or minimizing a matrixeffect of background materials of the bio-sample.

Among hindering factors which can be effectively reduced by the methodand the apparatus for measuring a concentration of an analyte in abio-sample according to an exemplary embodiment of the presentinvention, a representative example for a blood sample is hematocrit. Itis not necessary to improve a structure of an electrochemicalbio-sensor, i.e. a strip, or a reagent, and a structure of a measuringapparatus can also use a conventional circuit for measuring a currentvalue by applying a constant voltage.

Further, the method and the apparatus for measuring a concentration ofan analyte in a bio-sample according to an exemplary embodiment of thepresent invention do not modify a chronoamperometric section typicallyused in the conventional market and obtain a correction signal from arange of a perturbation potential to be subsequently applied. Therefore,it is possible to minimize a deviation with respect to hematocrit whilemaintaining conventional measuring performance and characteristics.

Also, the method and the apparatus for measuring a concentration of ananalyte in a bio-sample according to an exemplary embodiment of thepresent invention are capable of determining a concentration of ananalyte using a calibration equation obtained through multivariableregression analysis by comparing a function formed of features extractedfrom an induced current so as to be minimized in deviation with respectto hematocrit with the standard test results. Thus, an additionalprocess of obtaining hematocrit is not needed, and fluctuation inprecision which may occur during a process of obtaining two differentmeasurement values can be minimized.

The apparatus for measuring a concentration of an analyte in abio-sample according to an exemplary embodiment of the present inventionupgrades and inputs a program programmed using the calibration equationdetermined according to the method for measuring a concentration of ananalyte in a bio-sample according to an exemplary embodiment of thepresent invention. Thus, it is possible to obtain a concentration of ananalyte minimized in effect of hematocrit using a conventional strip andhardware as they are.

Further, the method for measuring a concentration of an analyte in abio-sample according to an exemplary embodiment of the present inventionis capable of more accurately determining a concentration of an analytethrough a more economical and efficient process as compared with aprocess in which hematocrit is obtained and then separately applied to acalibration equation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating a Λ-step ladder-type perturbationpotential used in a method for measuring a concentration of an analytein a bio-sample according to an exemplary embodiment of the presentinvention.

FIG. 2 is a graph illustrating an induced current obtained incorrespondence with the voltage applied as illustrated in FIG. 1.

FIG. 3 is a graph provided to explain a structure of a Λ-stepladder-type perturbation potential used in a method for measuring aconcentration of an analyte in a bio-sample according to an exemplaryembodiment of the present invention.

FIG. 4 provides front and rear perspective views of a measuringapparatus in which a calibration equation by means of a method formeasuring a concentration of an analyte in a bio-sample according to anexemplary embodiment of the present invention is stored.

FIG. 5 is a block diagram illustrating a circuit of the measuringapparatus for measuring a concentration of an analyte in a bio-sample asillustrated in FIG. 4.

FIG. 6 is a graph illustrating a correlation between a blood glucosemeasurement value measured by a measuring apparatus according tochronoamperometry and a YSI measurement value in a method for measuringa concentration of an analyte in a bio-sample according to a firstexemplary embodiment of the present invention.

FIG. 7 is a graph illustrating an effect of hematocrit on an averagevalue of blood glucose measurement values measured by a measuringapparatus according to chronoamperometry in the method for measuring aconcentration of an analyte in a bio-sample according to the firstexemplary embodiment of the present invention (a concentration of lessthan 100 mg/dL is expressed by an absolute error and a concentration of100 mg/dL or more is expressed by a relative error (%)).

FIG. 8 is a graph illustrating a correlation between a blood glucosemeasurement value obtained by using chronoamperometry and a stepladder-type perturbation potential and an YSI measurement value in amethod for measuring a concentration of an analyte in a bio-sampleaccording to a second exemplary embodiment of the present invention.

FIG. 9 is a graph illustrating an effect of hematocrit on an averagevalue of blood glucose measurement values obtained by usingchronoamperometry and a step ladder-type perturbation potential in themethod for measuring a concentration of an analyte in a bio-sampleaccording to the second exemplary embodiment of the present invention (aconcentration of less than 100 mg/dL is expressed by an absolute errorand a concentration of 100 mg/dL or more is expressed by a relativeerror (%)).

FIG. 10 is a flowchart illustrating a method for measuring aconcentration of an analyte in a bio-sample according to an exemplaryembodiment of the present invention.

FIG. 11 is a graph illustrating a correlation between a blood glucosemeasurement value obtained by using chronoamperometry, a stepladder-type perturbation potential, and a temperature value measured bya measuring apparatus and a YSI measurement value in a method formeasuring a concentration of an analyte in a bio-sample according to athird exemplary embodiment of the present invention (including samplesrespectively having a hematocrit value of 10, 20, 42, 55, and 70%).

FIG. 12 is a graph illustrating an effect of temperature on an averagevalue of blood glucose measurement values obtained by usingchronoamperometry, a step ladder-type perturbation potential, and atemperature value measured by a measuring apparatus in the method formeasuring a concentration of an analyte in a bio-sample according to thethird exemplary embodiment of the present invention (including sampleshaving a hematocrit value of 10, 20, 42, 55, and 70%; a concentration ofless than 100 mg/dL is expressed by an absolute error and aconcentration of 100 mg/dL or more is expressed by a relative error(%)).

FIG. 13 is a graph illustrating a correlation between a ketone bodymeasurement value obtained according to chronoamperometry and ameasurement value measured by reference equipment in a method formeasuring a concentration of an analyte in a bio-sample according to afourth exemplary embodiment of the present invention.

FIG. 14 is a graph illustrating an effect of hematocrit on an averagevalue of ketone body measurement values obtained according tochronoamperometry in the method for measuring a concentration of ananalyte in a bio-sample according to the fourth exemplary embodiment ofthe present invention (a concentration of less than 1.0 mmol/L isexpressed by an absolute error multiplied by 100 and a concentration of1.0 mmol/L or more is expressed by a relative error (%)).

FIG. 15 is a graph illustrating a correlation between a ketone bodymeasurement value obtained by using chronoamperometry and a stepladder-type perturbation potential and a measurement value measured by areference equipment in a method for measuring a concentration of ananalyte in a bio-sample according to a fifth exemplary embodiment of thepresent invention.

FIG. 16 is a graph illustrating an effect of hematocrit on an averagevalue of ketone body measurement values obtained by usingchronoamperometry and a step ladder-type perturbation potential in themethod for measuring a concentration of an analyte in a bio-sampleaccording to the fifth exemplary embodiment of the present invention (aconcentration of less than 1.0 mmol/L is expressed by an absolute errormultiplied by 100 and a concentration of 1.0 mmol/L or more is expressedby a relative error (%)).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Hereinafter, a method and an apparatus for measuring a concentration ofan analyte in a bio-sample according to exemplary embodiments of thepresent invention will be explained in detail with reference to theaccompanying drawings.

In the present specification, it will be explained that correcting ameasurement error generated due to hematocrit during measurement ofblood glucose is desirable in an exemplary embodiment. However, in thesame manner as a glucose test, by introducing a specific enzyme, it ispossible to correct concentration measurement values of variousmetabolites such as organic materials including β-hydroxybutyric acid,cholesterol, triglyceride, lactate, pyruvate, alcohol, bilirubin, uricacid, phenylketonuria, creatine, creatinine, glucose-6-phosphatedehydrogenase, and NAD(P)H, or inorganic materials.

Therefore, the present invention can be applied to quantification ofvarious metabolites by varying a kind of an enzyme included in a samplelayer composition.

For example, quantification of glucose, glutamate, cholesterol, lactate,ascorbic acid, alcohol, and bilirubin can be carried out using a glucoseoxidase (GOx), a glucose dehydrogenase (GDH), a glutamate oxidase, aglutamate dehydrogenase, a cholesterol oxidase, a cholesterol esterase,a lactate oxidase, an ascorbic acid oxidase, an alcohol oxidase, analcohol dehydrogenase, a bilirubin oxidase, and the like.

An electron transfer mediator which can be used together with theabove-described enzymes may be one of ferrocene, ruthenium hexamine(III)chloride, potassium ferricyanide, 1,10-phenanthroline-5,6-dione, andbipyridine, or an osmium complex including phenanthroline as a ligand,2,6-dimethyl-1,4-benzoquinone, 2,5-dichloro-1,4-benzoquinone,3,7-diamino-5-phenothiaziniumthionine, 1-methoxy-5-methylphenaziniummethylsulfate, methylene blue, and toluidine blue, but may not belimited to these compounds and may include organic and inorganicelectron transfer mediators capable of transferring an electron togetherwith an enzyme capable of catalyzing an oxidation/reduction reaction ofa metabolite.

As a portable measuring apparatus according to an exemplary embodimentof the present invention, a vis-a-vis electrochemical bio-sensor inwhich a working electrode and an counter electrode are provided ondifferent planes so as to face each other and a reagent compositionincluding an enzyme and an electron transfer mediator depending on amaterial is coated on the working electrode may be employed.

Further, as a portable measuring apparatus according to an exemplaryembodiment of the present invention, a planar electrochemical bio-sensorin which a working electrode and an counter electrode are provided onthe same plane and a reagent composition including an enzyme and anelectron transfer mediator depending on a material is coated on theworking electrode may be employed.

Hereinafter, a method and an apparatus for measuring a concentration ofan analyte in a bio-sample according to exemplary embodiments of thepresent invention will be explained in detail with reference to FIG. 1to FIG. 5.

FIG. 1 and FIG. 2 are graphs respectively illustrating a Λ-stepladder-type perturbation potential used in a method for measuring aconcentration of an analyte in a bio-sample and an induced currentobtained in correspondence with the voltage according to an exemplaryembodiment of the present invention, FIG. 3 is a graph provided toexplain a structure of a Λ-step ladder-type perturbation potential usedin a method for measuring a concentration of an analyte in a bio-sampleaccording to an exemplary embodiment of the present invention, FIG. 4provides front and rear perspective views of a measuring apparatus inwhich a calibration equation by means of a method for measuring aconcentration of an analyte in a bio-sample according to an exemplaryembodiment of the present invention is stored, and FIG. 5 is a blockdiagram illustrating a circuit of the measuring apparatus for measuringa concentration of an analyte in a bio-sample as illustrated in FIG. 4.

As illustrated in FIG. 1, in a method for measuring a concentration ofan analyte in a bio-sample according to an exemplary embodiment of thepresent invention, a step ladder-type perturbation potential is appliedconsecutively after a constant voltage (V_(DC)) is applied. Accordingly,an induced current is measured.

As illustrated in FIG. 3, a perturbation potential used in the methodfor measuring a concentration of an analyte in a bio-sample according tothe exemplary embodiment of the present invention is formed of a stepladder-type wave, the perturbation potential is characterized by aheight (V_(step)) of each step, an application time (t_(step)) for eachstep, a difference (V_(center)) between a middle voltage and a constantvoltage in the entire range of variations, a difference (V_(peak))between the middle voltage and a peak voltage, and a time difference(t_(cycle)) between a peak voltage of the entire step ladder-type waveand the peak voltage of the adjacent next step ladder-type wave, and hasa range as listed in the following Table 1.

Table 1 listing a range of a step ladder-type wave is just one exemplaryembodiment of the present invention, and can be modified and changed invarious ways depending on application.

TABLE 1 Minimum Appropriate Maximum value value value V_(step) 0.5 mV1-10 mV 20 mV t_(step) 0.001 s 0.01-0.05 s 0.1 s V_(DC) 50 mV 150-300 mV800 mV V_(center) −150 mV −100-100 mV 150 mV V_(peak) 5 mV 12-60 mV 150mV t_(cycle) 0.01 s 0.05-0.2 s 1 s

In the method for measuring a concentration of an analyte in abio-sample according to the exemplary embodiment of the presentinvention, current values used for determining a concentration of theanalyte are points which can be obtained from one step or a plurality ofsteps of a first or second induced current.

As illustrated in FIG. 4, a concentration measuring apparatus 100 formeasuring a concentration of an analyte in a bio-sample according to anexemplary embodiment of the present invention is capable of obtaining anadditional signal for correction within several seconds and preferablywithin 0.1 to 1 second by applying a perturbation potential that changesa potential while maintaining a pair of a working electrode and ancounter electrode of a conventional electrochemical bio-sensor, i.e., astrip 10.

As illustrated in FIG. 5, the concentration measuring apparatus 100 formeasuring a concentration of an analyte in a bio-sample according to theexemplary embodiment of the present invention is configured such that ifthe electrochemical bio-sensor 10 is provided at a connector 110, theconnector 110 is electrically connected to a current-voltage converter120, and a microcontroller (MCU) 150 can apply a perturbation potentialto the working electrode of the strip 10 without an additionalperturbation potential circuit through a digital-to-analog converter(DAC) circuit 130 provided at the concentration measuring apparatus 100to apply a constant voltage according to the conventionalchronoamperometry.

To do so, firmware of the concentration measuring apparatus 100 formeasuring a concentration of an analyte in a bio-sample according to theexemplary embodiment of the present invention stores a predeterminedconstant which can generate a perturbation potential in a memory of themeasuring apparatus 100, records a predetermined constant at a registerof the DAC circuit 130 when a constant voltage is applied, andincreases/decreases the constant stored in the memory at a predeterminedtime interval and records the constant at the register of the DACcircuit 130 when the perturbation potential is applied.

The microcontroller 150 applies an adequate voltage between the twoelectrodes of the strip depending on a constant recorded at the registerof the DAC circuit 130.

The first or second induced current measured by the strip 10 can bedirectly measured by an analog-to-digital converter (ADC) circuit 140through the connector 110 and the current-voltage converter 120.

When the perturbation potential is formed of a step form wave asillustrated in FIG. 3, a circuit can be simplified as compared with theother methods using AC or a linear scanning method, and it is possibleto reduce generation of a charging current which may be a hindrance toanalysis when pulses of various voltages are used.

As illustrated in FIG. 1, if a step ladder-type wave is applied at aregular interval at a constant amplitude right after a constant voltageis applied, a distribution of concentrations of components to beoxidized/reduced in a diffusion layer near the electrode fluctuates.

Such fluctuation or perturbation causes an important change incharacteristics of an induced current, and this change can be animportant means capable of removing or minimizing an effect ofhematocrit with current values obtained from one step or a plurality ofsteps constituting a step ladder-type wave.

Herein, an induced current is expressed as a first induced current or asecond induced current in order to show that they are different fromeach other due to a change in characteristics of the induced currentcaused by fluctuation or perturbation.

An application type of a step ladder-type perturbation potential havingperiodicity and additionally applied for a short time in order to removean effect of hematocrit from a calibration equation after a constantvoltage is applied will be referred to as a “Λ-step ladder-typeperturbation potential” or simply as a “step ladder potential”.

The above-described currents that are different from each other incharacteristics refer to currents which can be used as variables foreffectively separating or correcting an effect of hematocrit since theydifferently depend on blood glucose and hematocrit (hindering material).

For example, if two or more voltage pulses are applied at an appropriatetime interval and first and second induced currents are measured fromeach pulse, and values of the first and second induced currents aredetermined depending on blood glucose and hematocrit and thus can beexpressed by the following functions g₁ and g₂ of blood glucose andhematocrit.

If the blood glucose and the hematocrit contribute to the currents inthe same manner in the current functions g₁ and g₂ and a linear formulaof constants in the form of i₁=ki₂ is established, it will be said thatthe current have the same characteristics, and if not, it will be saidthat the currents have different characteristics.

As for the currents having the same characteristics, it is impossible toaccurately calculate an effect of hematocrit or it is difficult tocorrect an effect of hematocrit due to linear dependency among variablesduring regression analysis.

However, as for an induced current which can be obtained by applying astep ladder-type perturbation potential, a level of fluctuation in asample is continuously changed near an electrical double layer when eachstep moves up or down for a short time and an electron transfer speedand an effect of a charging current are changed accordingly. Thus, itmay be different from the current obtained according tochronoamperometry in characteristics.

As such, elements useful for forming a calibration equation used for themethod for measuring a concentration of an analyte in a bio-sampleaccording to an exemplary embodiment of the present invention due to agreat difference in characteristics between first and second inducedcurrents corresponding to a constant voltage and a perturbationpotential will be referred to as characteristic points, and non-modifiedcurrent values of the characteristic points or values appropriatelymodified to be variables suitable for a calibration equation will bereferred to as features.

In an electrochemical bio-sensor, an induced current obtained accordingto chronoamperometry can be approximated by the Cottrell equation when areagent of the bio-sensor is mixed with a sample in a sample cell andreaches a uniform liquid state.

${i(t)} = {\frac{{nFAD}^{\frac{1}{2}}C}{\pi \; t^{\frac{1}{2}}} = {{k(t)}{AD}^{\frac{1}{2}}C}}$

Herein, n denotes the number of electrons transferred per molecule of amaterial (for example, an electron transfer mediator) to beoxidized/reduced in an electrode, F denotes the Faraday constant, Adenotes an electrode area, D denotes a diffusion coefficient within asample of the material to be oxidized/reduced, and C denotes aconcentration of the material to be oxidized/reduced.

A characteristic point in a chronoamperometric section is a currentvalue at a point which is stably expressed by the Cottrell equationafter a constant voltage is applied. In the present electrochemicalbio-sensor, it is a time point with a lapse of time of several secondsto several minutes, preferably 1 to 10 seconds, after a constant voltageis applied.

As described above, the second induced current obtained from a stepladder-type perturbation potential is greatly different incharacteristics from the first induced current obtained when a constantvoltage is applied, and thus it can be used as a variable with highorthogonality in the whole calibration equation.

A method for finding characteristic points from second induced currentscorresponding to a section where the perturbation potential is appliedand a method for making features with these characteristic points are asfollows.

The following method is one of examples and can be modified and changedin various ways depending on a purpose of application.

1) Induced currents near peak and valley voltages of a specific stepladder type wave

2) The curvature of a curved line formed of induced currents of eachstep of the step ladder type wave

3) A difference between a current value of a peak and a current value ofa valley of the step ladder type wave

4) Induced currents in the middle of ups and downs of the step laddertype wave

5) Induced currents at a starting point and an ending point of each stepladder-type cycle

6) An average value of induced currents obtained from the stepladder-type wave

7) Values which can be obtained by expressing the current valuesobtained from the above features 1 to 6 by the four fundamentalarithmetic operations and mathematical functions such as an exponentialfunction, a logarithmic function, and a trigonometric function.

If characteristic points are found from the second induced currentscorresponding to the section where the perturbation potential is appliedand features are made with current values obtained from thesecharacteristic points and multivariable regression analysis is appliedto a linear mixture thereof, it is possible to obtain a calibrationequation minimized in effect of hematocrit.

A specific method for making a calibration equation minimized in effectof hematocrit will be explained in detail with reference to thefollowing first to fifth exemplary embodiments.

However, the calibration equation to which multivariable regressionanalysis is applied by linearly mixing the features may be greatlychanged depending on a material of the electrodes used in theelectrochemical bio-sensor, arrangement of the electrodes, a shape of aflow path, and a characteristic of a reagent to be used.

The calibration equation used for the method for measuring aconcentration of an analyte in a bio-sample according to an exemplaryembodiment of the present invention can be applied to a generalelectrochemical bio-sensor having a sample cell including a pair of aworking electrode and an counter electrode. Particularly, if a materialto be measured is glucose or a ketone body in blood or a metabolitewhich can be electrochemically measured, it is useful for analysis of,for example, creatine, lactate, cholesterol, phenylketonuria,glucose-6-phosphate dehydrogenase, and the like.

In order to perform the method of the present invention for measuring aconcentration of an analyte in a bio-sample according to an exemplaryembodiment of the present invention, a feature function formed usingfeatures which can be obtained from first and second induced currentscorresponding to a constant voltage and a step ladder-type perturbationpotential needs to be optimized by multivariable regression analysis soas to minimize a deviation of hematocrit through experiments usingsamples under various conditions, and a calibration equation needs to bedeveloped.

Then, the calibration equation is realized by firmware of the measuringapparatus and thus can be used when a blood sample is analyzed.

[First Exemplary Embodiment] Method for Measuring Blood Glucose usingInduced Current Corresponding to Constant Voltage

A sample cell of an electrochemical bio-sensor used for a method formeasuring a concentration of an analyte in a bio-sample according to afirst exemplary embodiment of the present invention is a disposablestrip formed of two screen-printed carbon electrodes, and the electrodesare coated with a glucose dehydrogenase and an electron transfermediator (thionine, ruthenium hexamine chloride).

The measuring apparatus 100 used for the method for measuring aconcentration of an analyte in a bio-sample according to the firstexemplary embodiment of the present invention is commercially availableCareSens N (brand name) as illustrated in FIG. 4.

In the method for measuring a concentration of an analyte in abio-sample according to the first exemplary embodiment of the presentinvention, firmware of the measuring apparatus 100 is used as it is toobtain a first induced current corresponding to a constant voltage byapplying the constant voltage from the microcontroller 150 to theworking electrode through the digital-to-analog converter circuit 130,and a blood glucose value is calculated.

The experiment was conducted at a temperature of 23° C., and YSIequipment was used as reference equipment.

In order to check a deviation caused by hematocrit, blood experimentscan be conducted as follows.

Blood collected from venous blood is divided into red blood cells andplasma through centrifugation, and the red blood cells and the plasmaare mixed again at an appropriate ratio so as to obtain a desiredhematocrit. Then, samples respectively having hematocrit values of 10,20, 30, 42, 50, 60, and 70% are prepared. A glucose concentration isprepared by adding a glucose solution having a high concentration toeach sample.

The blood samples are prepared such that blood glucose values can beapproximately 30, 80, 130, 200, 350, 450, and 600 mg/dL with respect tothe respective hematocrit values, and an actual blood glucose value ofeach sample is measured by reference equipment to be determined.

Meanwhile, the measuring apparatus 100 records a first induced currentcorresponding to a constant voltage according to the conventionalchronoamperometry.

A voltage applied at this time is 0 V when being applied between the twocarbon electrodes for 3 seconds after inflow of blood, and is 200 mVwhen being applied between the two carbon electrodes for 2 secondsthereafter. Therefore, current values after the lapse of 5 seconds arerecorded with respect to the respective samples.

A blood glucose measurement formula is made based on the sample having ahematocrit value of 42%. The blood glucose measurement formula is asfollows.

Glucose=slope*i _(t=5s) (a current value after the lapse of 5seconds)+intercept

A blood glucose calibration equation is determined by calculating aslope and an intercept from the experimental data by the least squaresmethod.

The results of calculation with respect to all hematocrit samples usingthe calibration equation obtained as such are as illustrated in FIG. 6and FIG. 7.

FIG. 6 is a graph illustrating a correlation between a blood glucosemeasurement value measured by a measuring apparatus according tochronoamperometry and a YSI measurement value in a method for measuringa concentration of an analyte in a bio-sample according to a firstexemplary embodiment of the present invention, and FIG. 7 is a graphillustrating an effect of hematocrit on an average value of bloodglucose measurement values measured by a measuring apparatus accordingto chronoamperometry in the method for measuring a concentration of ananalyte in a bio-sample according to the first exemplary embodiment ofthe present invention (a concentration of less than 100 mg/dL isexpressed by an absolute error and a concentration of 100 mg/dL or moreis expressed by a relative error (%)).

As illustrated in FIG. 6 and FIG. 7, in the method for measuring aconcentration of an analyte in a bio-sample according to the firstexemplary embodiment of the present invention, the average value ofblood glucose measurement values measured by the measuring apparatusaccording to chronoamperometry maintains linearity with all hematocritvalues. However, it can be confirmed that as hematocrit increases, aslope decreases.

Particularly, as illustrated in FIG. 7, it can be seen that, for atendency of the blood glucose measurement values with respect to therespective hematocrit values, a deviation increases toward both endsbased on 42%.

[Second Exemplary Embodiment] Example of Calibration Equation usingFeatures Extracted from Characteristic Points after Application ofConstant Voltage and Perturbation Potential

With the strip 10 and the measuring apparatus 100 used for the methodfor measuring a concentration of an analyte in a bio-sample according tothe first exemplary embodiment of the present invention, a calibrationequation minimized in effect of hematocrit can be obtained.

The experimental environment and samples used for a method for measuringa concentration of an analyte in a bio-sample according to a secondexemplary embodiment of the present invention are the same as those ofthe first exemplary embodiment of the present invention.

A concentration measuring apparatus 100 for measuring a concentration ofan analyte in a bio-sample according to a second exemplary embodiment ofthe present invention is different from the blood glucose measuringapparatus 100 according to the first exemplary embodiment in applicationof a voltage.

In the concentration measuring apparatus 100 for measuring aconcentration of an analyte in a bio-sample according to a secondexemplary embodiment of the present invention, firmware of the measuringapparatus 100 is modified as follows such that an appropriateperturbation potential can be applied right after a conventionalconstant voltage is applied.

The firmware of the concentration measuring apparatus 100 for measuringa concentration of an analyte in a bio-sample according to a secondexemplary embodiment of the present invention stores a predeterminedconstant which can generate a perturbation potential in a memory of themeasuring apparatus 100, records a predetermined constant at a registerof the DAC circuit when a constant voltage is applied, andincreases/decreases the constant stored in the memory at a predeterminedtime interval and records the constant at the register of the DACcircuit when the perturbation potential is applied.

An adequate voltage is applied between the two electrodes of the stripdepending on a constant recorded at the register of the DAC circuit.

A structure of a step ladder-type perturbation potential applied as suchis described in the following Table 2.

TABLE 2 V_(step) 2.0 mV t_(step) 0.0025 s V_(DC) 200 mV V_(center) 200mV V_(peak) 20 mV t_(cycle) 0.1 s

The prepared samples are measured by the blood glucose measuringapparatus 100 prepared as such. Induced currents obtained from themeasurement are stored in a computer.

Features are formed of optimum characteristic points extracted byanalyzing the stored data by a blood glucose formula, and a calibrationequation formed of these features is formed. Then, a coefficient of eachfeature is determined through multivariable regression analysis so as tocomplete the calibration equation. The calibration equation is asfollows.

${glucose}{= {\sum\limits_{j}{c_{j}{f_{j}\left( {i,\ T} \right)}}}}$

Herein, i denotes one or more current values which can be obtained fromthe first induced current and the second induced current, and thefeatures used herein are as follows.

f₁=i at 5 s (an induced current corresponding to a constant voltage)

f₂=i at 5.4925 s (an induced current at a point of an ascending step ofthe sixth step ladder type potential)

f₃=i at 5.4425 s (an induced current at a point of a descending step ofthe fifth step ladder type potential)

f₄=curvature (the curvature formed of induced currents at descendingsteps of the fifth step ladder potential)

f₅=f₁ ²

f₆=f₂ ²

f₇=f₃ ²

f₈=f₄ ²

f₉=1/f₁

f₁₀=1/f₂

f₁₁=1/f₃

f₁₂=1/f₄

A model formed of the above-described features is established, and inorder to match blood glucose values calculated with respect to therespective samples with values measured by the YSI under varioushematocrit conditions, the measuring apparatus used in the firstexemplary embodiment adds a weighted value with respect to the standardhematocrit of 42% so as to be close to a concentration obtainedaccording to chronoamperometry only, and optimizes the coefficients ofthe respective features by multivariable regression analysis. A newcalibration equation obtained as such can minimize an effect of ahindering material while maintaining the conventional calibration methodaccording to chronoamperometry.

The calibration equation is stored in the measuring apparatus togetherwith firmware modified so as to apply a perturbation potential afterapplication of a constant voltage. The results according to the newcalibration equation are as illustrated in FIG. 8 and FIG. 9.

FIG. 8 is a graph illustrating a correlation between a blood glucosemeasurement value obtained by using chronoamperometry and a stepladder-type perturbation potential and a YSI measurement value in themethod for measuring a concentration of an analyte in a bio-sampleaccording to the second exemplary embodiment of the present invention,and FIG. 9 is a graph illustrating an effect of hematocrit on an averagevalue of blood glucose measurement values obtained by usingchronoamperometry and a step ladder-type perturbation potential in themethod for measuring a concentration of an analyte in a bio-sampleaccording to the second exemplary embodiment of the present invention (aconcentration of less than 100 mg/dL is expressed by an absolute errorand a concentration of 100 mg/dL or more is expressed by a relativeerror (%)).

As can be seen from FIG. 8, the blood glucose measurement value obtainedby using chronoamperometry and a step ladder-type perturbation potentialand the YSI measurement value have a very close correlation, and as canbe seen from FIG. 9, the effect of hematocrit on the average value ofblood glucose measurement values obtained by using chronoamperometry anda step ladder-type perturbation potential decreases to less than about±5%.

A method for measuring a concentration of an analyte in a bio-sampleaccording to an exemplary embodiment of the present invention will beexplained with reference to FIG. 10.

FIG. 10 is a flowchart illustrating a method for measuring aconcentration of an analyte in a bio-sample according to an exemplaryembodiment of the present invention.

As illustrated in FIG. 10, a method for measuring a concentration of ananalyte in a bio-sample according to an exemplary embodiment of thepresent invention includes a step S110 of introducing a liquidbio-sample into a sample cell in which an oxidation/reduction enzymecapable of catalyzing an oxidation/reduction reaction of the analyte andan electron transfer mediator are fixed and a working electrode and ancounter electrode are provided, a step S120 of obtaining a first inducedcurrent by applying a constant DC voltage to the working electrode tostart the oxidation/reduction reaction of the analyte and proceed withan electron transfer reaction, a step S130 of obtaining a second inducedcurrent by applying a Λ-step ladder-type perturbation potential afterapplying the constant DC voltage, a step S140 of calculating apredetermined feature from two or more characteristic points from thefirst induced current or the second induced current, and a step S150 ofcalculating a concentration of the analyte using a calibration equationformed of at least one feature function so as to minimize an effect ofat least two hindering materials in the bio-sample.

After the constant DC voltage is applied, the Λ-step ladder-typeperturbation potential is applied in the form of a step form wave byusing a conventional DAC circuit as described above.

The step S140 of calculating a predetermined feature from the firstinduced current or the second induced current includes obtaining afeature from a current value at a predetermined characteristic point ofthe first induced current or the second induced current or by modifyingthe current value.

[Third Exemplary Embodiment] Example of Calibration Equation forCalculating Accurate Blood Glucose Value in Various Temperature Rangesusing Temperature as Additional Feature

With the strip and the measuring apparatus used for the method formeasuring a concentration of an analyte in a bio-sample according to thesecond exemplary embodiment of the present invention, a calibrationequation minimized in effect of a temperature and hematocrit can beobtained.

The experimental environment and samples used herein are similar tothose used for the method for measuring a concentration of an analyte ina bio-sample according to the second exemplary embodiment of the presentinvention.

That is, samples respectively having a hematocrit value of 10, 20, 42,55, and 70% and a blood glucose concentration of 50, 130, 250, 400, and600 mg/dL were prepared, and experiments were respectively conducted at5, 12, 18, 23, 33, and 43° C.

A measuring apparatus 100 used for a method for measuring aconcentration of an analyte in a bio-sample according to the thirdexemplary embodiment of the present invention is modified from the bloodglucose measuring apparatus used in the second exemplary embodiment inapplication of a voltage.

A structure of a step ladder-type perturbation potential used for themethod for measuring a concentration of an analyte in a bio-sampleaccording to the third exemplary embodiment of the present invention isdescribed in the following Table 3.

TABLE 3 V_(step) 2.0 mV t_(step) 0.005 s V_(DC) 200 mV V_(center) 200 mVV_(peak) 20 mV t_(cycle) 0.2 s

The prepared samples are measured at each temperature by the measuringapparatus 100 prepared as such. Induced currents obtained from themeasurement are stored in a computer.

Features are formed of optimum characteristic points extracted byanalyzing the stored data by a blood glucose formula, and a calibrationequation formed of these features is formed. Then, a coefficient of eachfeature is determined through multivariable regression analysis so as tocomplete the calibration equation. The calibration equation is asfollows.

${{ketone}\mspace{14mu} {body}} = {\sum\limits_{j}{c_{j}{f_{j}(i)}}}$

Herein, i denotes one or more current values which can be obtained fromthe first induced current and the second induced current, T denotestemperature values that are independently measured, and the featuresused herein are as follows.

f₁=i at 5 s (an induced current corresponding to a constant voltage)

f₂=i at 5.2675 s (an induced current at a point descending from a peakof the second step ladder type potential)

f₃=i at 5.3675 s (an induced current at a point ascending from a valleyof the third step ladder type potential)

f₄=curvature (the curvature formed of induced currents at descendingsteps of the second step ladder potential)

f₅=Peak-to-Peak (a difference between a peak voltage and a valleyvoltage of the second step ladder potential)

f₆=f₁ ²

f₇=f₂ ²

f₈=f₃ ²

f₉=f₄ ²

f₁₀=f₅ ²

f₁₁=1/f₁

f₁₂=1/f₄

f₁₃=T

f₁₄=T²

f₁₅=f₁*T

A model formed of the above-described features is established, and asexplained in the second exemplary embodiment of the present invention,the coefficients of the respective features are optimized bymultivariable regression analysis based on the blood glucose valuesmeasured by the reference equipment YSI.

A calibration equation obtained as such is stored in the measuringapparatus together with firmware modified so as to apply a perturbationpotential after application of a constant voltage in the same manner asthe second exemplary embodiment. The results according to the newcalibration equation are as illustrated in FIG. 11 and FIG. 12.

FIG. 11 is a graph illustrating a correlation between a blood glucosemeasurement value obtained by using chronoamperometry, a stepladder-type perturbation potential, and a temperature value measured bya measuring apparatus and a YSI measurement value in the method formeasuring a concentration of an analyte in a bio-sample according to thethird exemplary embodiment of the present invention (including samplesrespectively having a hematocrit value of 10, 20, 42, 55, and 70%).

FIG. 12 is a graph illustrating an effect of temperature on an averagevalue of blood glucose measurement values obtained by usingchronoamperometry, a step ladder-type perturbation potential, and atemperature value measured by a measuring apparatus in the method formeasuring a concentration of an analyte in a bio-sample according to thethird exemplary embodiment of the present invention (a concentration ofless than 100 mg/dL is expressed by an absolute error and aconcentration of 100 mg/dL or more is expressed by a relative error(%)).

As illustrated in FIG. 10, measurement of a blood glucose value includesintroducing blood, applying a constant voltage, applying a stepladder-type perturbation potential, calculating features from inducedcurrents, and obtaining an accurate blood glucose value using the newcalibration equation.

[Fourth Exemplary Embodiment] Example of Calibration Equation forMeasuring Ketone Body

In a method for measuring a concentration of an analyte in a bio-sampleaccording to a fourth exemplary embodiment of the present invention, asample cell of the electrochemical bio-sensor 10 is a disposable stripformed of two screen-printed carbon electrodes, and if the electrodesare coated with a ketone body dehydrogenase and an electron transfermediator (1-methoxy-5-methylphenazinium methyl sulfate, rutheniumhexamine chloride), an induced current is obtained by applying aconstant voltage at 23° C. and a ketone body concentration iscalculated.

Blood experiments for checking a deviation caused by hematocrit areconducted similarly to the first exemplary embodiment. Blood samplesrespectively having a hematocrit value of 20, 30, 42, 50, 60, and 70%are prepared.

The blood samples are prepared such that ketone body concentrations canbe approximately 0.1, 0.5, 1, 2, 3, 4.2, and 5 mmol/L with respect tothe respective hematocrit values, and an actual blood glucose value ofeach sample is measured by reference equipment (RX Monaco, Randox) andthen determined.

Meanwhile, the measuring apparatus having the same structure as theblood glucose measuring apparatus used in the above-described exemplaryembodiment records an induced current corresponding to a constantvoltage.

An applied voltage used herein is 200 mV when being applied between thetwo electrodes within the strip for 4 seconds after inflow of blood, is0 mV when being applied for 4 seconds thereafter, and then is 200 mVwhen being applied for 2 seconds thereafter.

Current values after the lapse of 10 seconds are recorded with respectto the respective samples.

A ketone body measurement formula is made based on the sample having ahematocrit of 42%.

The ketone body measurement formula is as follows.

Ketone Body=slope*i _(t=10s) (a current value after the lapse of 10seconds)+intercept

A calibration equation is obtained by calculating a slope and anintercept from the experimental data by the least square methodexperimental data.

The results of calculation with respect to all hematocrit samples usingthe ketone body calibration equation obtained as such are as illustratedin FIG. 13 and FIG. 14.

FIG. 13 is a graph illustrating a correlation between a ketone bodymeasurement value obtained according to chronoamperometry and ameasurement value measured by reference equipment in the method formeasuring a concentration of an analyte in a bio-sample according to thefourth exemplary embodiment of the present invention, and FIG. 14 is agraph illustrating an effect of hematocrit on an average value of ketonebody measurement values obtained according to chronoamperometry in themethod for measuring a concentration of an analyte in a bio-sampleaccording to the fourth exemplary embodiment of the present invention (aconcentration of less than 1.0 mmol/L is expressed by an absolute errormultiplied by 100 and a concentration of 1.0 mmol/L or more is expressedby a relative error (%)).

As illustrated in FIG. 13 and FIG. 14, it can be confirmed that theaverage value of ketone body measurement values obtained according tochronoamperometry has a decreasing slope as hematocrit increases.

Further, as illustrated in FIG. 14, it can be seen that, for a tendencyof the ketone body measurement values with respect to the respectivehematocrit values according to chronoamperometry, a deviation increasestoward both ends based on 42%.

[Fifth Exemplary Embodiment] Example of Calibration Equation forMeasuring Ketone Body using Features Extracted from CharacteristicPoints After Application of Constant Voltage and Perturbation Potential

With the strip and the measuring apparatus used for the method formeasuring a concentration of an analyte in a bio-sample according to thefourth exemplary embodiment of the present invention, a calibrationequation for measuring a ketone body using features extracted fromcharacteristic points after application of a constant voltage and aperturbation potential can be obtained.

The experimental environment and samples used herein are the same asthose used for the method for measuring a concentration of an analyte ina bio-sample according to the fourth exemplary embodiment of the presentinvention.

A measuring apparatus is different from the measuring apparatus used inthe fourth exemplary embodiment in application of a voltage. That is,firmware of the measuring apparatus is modified such that a perturbationpotential described in the following table can be applied right after aconventional constant voltage is applied.

In a method for measuring a concentration of an analyte in a bio-sampleaccording to the fifth exemplary embodiment of the present invention, avoltage is applied in the form of a step ladder-type perturbationpotential described in the following Table 4 right after the voltageused in the fourth exemplary embodiment.

TABLE 4 V_(step) 1.5 mV t_(step) 0.0025 s V_(DC) 200 mV V_(center) 250mV V_(peak) 15 mV t_(cycle) 0.1 s

The prepared samples are measured by the measuring apparatus prepared assuch. Induced currents obtained from the measurement are stored in acomputer.

Features are formed of optimum characteristic points extracted byanalyzing the stored data by a blood glucose formula, and a calibrationequation formed of these features is formed. Then, a coefficient of eachfeature is determined through multivariable regression analysis so as tocomplete the calibration equation.

The calibration equation for measuring a ketone body is as follows.

${{ketone}\mspace{14mu} {body}} = {\sum\limits_{j}{c_{j}{f_{j}(i)}}}$

Herein, i denotes one or more current values which can be obtained fromthe first induced current and the second induced current, and thefeatures used herein are as follows.

f₁=current at 10 s (an induced current corresponding to a constantvoltage)

f₂=current at 8.12 s (an initial induced current corresponding to aconstant voltage)

f₃=current at 10.27 s (an induced current at a voltage near a valley ofthe third step ladder type potential)

f₄=current at 10.4925 s (an induced current at a voltage near a valleyof the fifth step ladder type potential)

f₅=curvature (the curvature formed of induced currents at descendingsteps of the fifth step ladder potential)

f₆=f₁ ²

f₇=f₂ ²

f₈=f₃ ²

f₉=f₄ ²

f₁₀=f₅ ²

f₁₁=1/f₁

f₁₂=1/f₅

A model formed of the above-described features is established, and inorder to match blood glucose values calculated with respect to therespective samples with values measured by the reference equipment undervarious hematocrit conditions, a weighted value is added with respect tothe standard hematocrit of 42% so as to be close to a concentrationobtained according to chronoamperometry only, and the coefficients ofthe respective features are optimized by multivariable regressionanalysis.

A calibration equation obtained as such is stored in the measuringapparatus together with firmware modified so as to apply a perturbationpotential after application of a constant voltage. The results accordingto the calibration equation are as illustrated in FIG. 15 and FIG. 16.

FIG. 15 is a graph illustrating a correlation between a ketone bodymeasurement value obtained by using chronoamperometry and a stepladder-type perturbation potential and a measurement value measured by areference equipment in the method for measuring a concentration of ananalyte in a bio-sample according to the fifth exemplary embodiment ofthe present invention, and FIG. 16 is a graph illustrating an effect ofhematocrit on an average value of ketone body measurement valuesobtained by using chronoamperometry and a step ladder-type perturbationpotential in the method for measuring a concentration of an analyte in abio-sample according to the fifth exemplary embodiment of the presentinvention (a concentration of less than 1.0 mmol/L is expressed by anabsolute error multiplied by 100 and a concentration of 1.0 mmol/L ormore is expressed by a relative error (%)).

The effect of the method for measuring a concentration of an analyte ina bio-sample according to the exemplary embodiments of the presentinvention can be clearly seen by comparing the first exemplaryembodiment with the second exemplary embodiment of the present inventionand comparing the third exemplary embodiment with the fourth exemplaryembodiment.

That is, it is possible to directly obtain a result that is minimized inmatrix effect of a hindering factor such as hematocrit from calibrationequation, without using an additional correction formula, by using aconventional bio-sensor in a measuring apparatus according to thegenerally-used chronoamperometry and adding a step ladder-typeperturbation potential (FIG. 1) to a conventional voltage applicationmethod only for a short time.

Further, as can be seen from the third exemplary embodiment of thepresent invention, if a calibration equation is obtained using atemperature measured by a measuring apparatus as an additional feature,it is possible to obtain a measurement result that is minimized in bothof a matrix effect and a temperature effect by a simple calculation.

While this invention has been described in connection with what ispresently considered to be practical exemplary embodiments, it is to beunderstood that the invention is not limited to the disclosedembodiments, but, on the contrary, is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

Description of Symbols

10: Electrochemical bio-sensor 100: Measuring apparatus (Strip) 110:Connector 120: Current-voltage converter 130: DAC circuit 140: ADCcircuit 150: Microcontroller

What is claimed is:
 1. An apparatus for measuring a concentration of ananalyte in a bio-sample, the apparatus comprising: a connector to whicha sample cell in which an oxidation/reduction enzyme capable ofcatalyzing an oxidation/reduction reaction of the analyte and anelectron transfer mediator are fixed and a working electrode and ancounter electrode are provided is for being inserted; adigital-to-analog converter circuit configured to apply a constant DCvoltage to start the oxidation/reduction reaction of the analyte,proceed with an electron transfer reaction, and apply a Λ-stepladder-type perturbation potential for fluctuating a potential of thesample cell after applying the constant DC voltage; and amicrocontroller configured to control the digital-to-analog convertercircuit and directly obtain a concentration value of the analyte from acalibration equation using the Λ-step ladder-type perturbationpotential, wherein the calibration equation is formed of at least onefeature function so as to minimize an effect of at least one hinderingmaterial in the bio-sample, wherein the Λ-step ladder-type perturbationpotential is characterized by a height (V_(step)) of each step, anapplication time (t_(step)) for each step, a difference (V_(center))between a middle voltage and a constant voltage in an entire range ofvariations, a difference (V_(peak)) between a middle voltage and a peakvoltage, and a time difference (t_(cycle)) between a peak voltage of anentire step ladder-type wave and a peak voltage of an adjacent next stepladder-type wave, wherein the digital-to-analog converter circuit,linked with the microcontroller, is configured to apply the constant DCvoltage and the Λ-step ladder-type perturbation potential to the workingelectrode, and wherein the apparatus is configured to select acharacteristic point, having different linearity with respect to theanalyte and the hindering material, from a first or second inducedcurrent, configured to form a feature of the characteristic point, andconfigured to form the calibration equation of the feature.
 2. Theapparatus for measuring a concentration of an analyte in a bio-sample ofclaim 1, wherein the microcontroller is configured to store apredetermined constant value which can generate the Λ-step ladder-typeperturbation potential, configured to record a predetermined constant ata register of the digital-to-analog converter circuit when a constantvoltage is applied, and configured to increase/decrease the constant ata predetermined time interval and configured to record the constant atthe register of the digital-to-analog converter circuit when the Λ-stepladder-type perturbation potential is applied, and the digital-to-analogconverter circuit is configured to apply the constant voltage or theΛ-step ladder-type perturbation potential between the working electrodeand the counter electrode depending on a constant value recorded at adigital-to-analog circuit register.
 3. The apparatus for measuring aconcentration of an analyte in a bio-sample of claim 1, wherein theapparatus is configured to obtain the second induced current within 0.1to 1 second after the first induced current is obtained.
 4. Theapparatus for measuring a concentration of an analyte in a bio-sample ofclaim 3, wherein the apparatus is configured to form the feature of thecharacteristic point by using one of second induced currents near peakand valley voltages of a specific step ladder type, curvature of acurved line formed of induced currents of each step of the Λ-stepladder-type perturbation potential, a difference between a current valueof a peak and a current value of a valley of the Λ-step ladder-typeperturbation potential, induced currents in a middle of ups and downs ofthe Λ-step ladder-type perturbation potential, induced currents at astarting point and an ending point of each step ladder-type perturbationpotential cycle, and an average value of induced currents obtained fromthe Λ-step ladder-type perturbation potential, and the apparatus isconfigured to use values which can be obtained by expressing the currentvalues obtained therefrom by four fundamental arithmetic operations andmathematical functions including an exponential function, a logarithmicfunction, and a trigonometric function.
 5. The apparatus for measuring aconcentration of an analyte in a bio-sample of claim 1, wherein theapparatus is configured to obtain the calibration equation by applyingmultivariable regression analysis to the feature function as a linearmixture of the feature, and the calibration equation is variabledepending on a material of the working electrode and the counterelectrode, an arrangement of the working electrode and the counterelectrode, a shape of a flow path, and a characteristic of a reagent tobe used.
 6. The apparatus for measuring a concentration of an analyte ina bio-sample of claim 5, wherein the analyte is one of glucose,β-hydroxybutyric acid, cholesterol, triglyceride, lactate, pyruvate,alcohol, bilirubin, uric acid, phenylketonuria, creatine, creatinine,glucose-6-phosphate dehydrogenase, NAD(P)H, and a ketone body.
 7. Theapparatus for measuring a concentration of an analyte in a bio-sample ofclaim 6, wherein the oxidation/reduction enzyme is one of a glucoseoxidase (GOx), a glucose dehydrogenase (GDH), a glutamate oxidase, aglutamate dehydrogenase, a cholesterol oxidase, a cholesterol esterase,a lactate oxidase, an ascorbic acid oxidase, an alcohol oxidase, analcohol dehydrogenase, a bilirubin oxidase, and a ketone bodydehydrogenase.
 8. The apparatus for measuring a concentration of ananalyte in a bio-sample of claim 1, wherein the electron transfermediator which can be used together with the oxidation/reduction enzymeis one of ferrocene, ruthenium hexamine(III) chloride, potassiumferricyanide, 1,10-phenanthroline-5,6-dione, and bipyridine, or anosmium complex including phenanthroline as a ligand,2,6-dimethyl-1,4-benzoquinone, 2,5-dichloro-1,4-benzoquinone,3,7-diamino-5-phenothiaziniumthionine, 1-methoxy-5-methylphenaziniummethylsulfate, methylene blue, and toluidine blue.
 9. The apparatus formeasuring a concentration of an analyte in a bio-sample of claim 1,wherein the digital-to-analog converter circuit is configured to applythe constant DC voltage, having a voltage range of 0 to 800 mV,consecutively or intermittently for 1 second or more to less than 1minute, and the apparatus is configured to measure the first inducedcurrent one time or several times while the constant DC voltage isapplied.
 10. The apparatus for measuring a concentration of an analytein a bio-sample of claim 1, wherein the height (V_(step)) of a step ofthe Λ-step ladder-type perturbation potential is 0.5 to 20 mV, aduration time (t_(step)) of the step is 0.001 to 0.1 second, thedifference (V_(center)) between the middle voltage and the constantvoltage in the entire range of variations is −150 to 150 mV, thedifference (V_(peak)) between the middle voltage and the peak voltage ofthe Λ-step ladder-type perturbation potential is 5 to 150 mV, and acycle of the Λ-step ladder-type perturbation potential or the timedifference (t_(cycle)) between the peak voltage of the entire stepladder-type wave and the peak voltage of the adjacent next stepladder-type wave is in a range of 0.01 to 1 second.
 11. The apparatusfor measuring a concentration of an analyte in a bio-sample of claim 1,wherein the feature function includes a function using an inducedcurrent value obtained from the constant DC voltage, a function using aninduced current value obtained from the Λ-step ladder-type perturbationpotential, a function using a temperature value measured by theapparatus, and a function which can be obtained by expressing measuredcurrent values by four fundamental arithmetic operations andmathematical functions including an exponential function, a logarithmicfunction, and a trigonometric function.
 12. The apparatus for measuringa concentration of an analyte in a bio-sample of claim 1, wherein thecalibration equation is one of glucose=Σ_(j) c_(j)f_(j)(i),glucose=Σ_(j) c_(j)f_(j)(i, T), and ketone body=Σ_(j) c_(j)f_(j)(i),wherein i denotes one or more current values which can be obtained fromthe first induced current and the second induced current, and T denotestemperature values that are independently measured.